Volume 19, Issue 5, Pages 622-632 (May 2011) Structure and Activity of a Novel Archaeal β-CASP Protein with N-Terminal KH Domains  Ana P.G. Silva, Maria Chechik, Robert T. Byrne, David G. Waterman, C. Leong Ng, Eleanor J. Dodson, Eugene V. Koonin, Alfred A. Antson, Callum Smits  Structure  Volume 19, Issue 5, Pages 622-632 (May 2011) DOI: 10.1016/j.str.2011.03.002 Copyright © 2011 Elsevier Ltd Terms and Conditions

Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Sequence Alignment of MTH1203 and Its Archaeal Orthologs with Human CPSF-73 and Thermus thermophilus TTHA0252 The GXXG motif of the KH fold is highlighted by a red box. The conserved motifs of β-CASP proteins are labeled beneath the consensus. The secondary structure assignment and domain structure is that of the MTH1203. Sequences were aligned using ClustalW (Thompson et al., 1994) and formatted with Aline (Bond and Schuttelkopf, 2009). Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Structure of MTH1203 and Its Homology with β-CASP Proteins from Different Kingdoms (A) Domain organization. Two N-terminal domains with a KH fold (blue and cyan) are connected by a linker segment (orange) with the central metallo-β-lactamase domain (green) followed by the β-CASP clamp domain (yellow). (B) Ribbon diagram of MTH1203 (PDB ID 2ycb) shown in stereo and colored as in (A). (C) Ribbon diagrams of the human CPSF-73 (PDB ID 2i7t) and the bacterial RNase TTHA0252 (PDB ID 2dkf). Red spheres correspond to Zn ions; a phosphate molecule in MTH1203 and a sulfate molecule in CPSF-73 are shown in sticks. The colors of the domains are the same as in (A). See also Figure S1. Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Metal-Containing Active Site of the MBL Domain (A) Anomalous difference maps calculated using the peak data collected at the zinc absorption edge and contoured at 5 σ. (B) Superposition of the MBL active sites of MTH1203 (green), human CPSF-73 (cyan), and bacterial TTHA0252 (magenta). The spheres correspond to the Zn atoms while the MTH1203 phosphate and the CPSF-73 sulfate molecules are shown in orange and yellow sticks, respectively. Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 KH Domains: Structures and Functional Implications (A) Superposition of the KHa (blue) and KHb (cyan) domains. (B) The KHb domain, rotated 180° around a vertical axis with respect to (A), is superposed with the Poly(C)-Binding Protein-2 KH1 domain (PCBP2-KH1, orange) complexed with 5′-AACCCU-3′ RNA segment (PDB ID 2py9; black). The G-loop of both KH domains and the variable loop (V-loop) of the PCBP2-KH1 are labeled. The side chains of K31 and K32 of PCBP-2 as well as K115 and Y116 of MTH1203 are highlighted. (C) Models of MTH1203 KHa and KHb domains in complex with RNA segments (black) generated by superposition with the PCBP2-RNA complex. (D) Sequence alignment of the MTH1203 KHa and KHb domains. The black box highlights the conserved GXXG RNA-binding motif found in the KHb of MTH1203. Side chains of the Lys and Tyr residues of this loop are shown in (A). Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 MTH1203 Binds Single Stranded U-Rich Sequences The change in fluorescence polarization anisotropy of fluoresceine-labeled RNAs was measured using increasing MTH1203 concentrations with BSA as a negative control. (A) Titrations performed using the degenerate sequence 5′-AAXXXXGG-3′. (B) Titration of U7, U4C4 and C4U4 indicated an interaction with a calculated Kd of 9.2, 3.6, and 5.2 μM, respectively. Calculation of the dissociation constants using Scientist (Micromath) indicated a good fit of the data, with the associated residual errors of 4% or less (data not shown). Data points correspond to multiple measurements from the same titration. See also Figures S2 and S3. Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 MTH1203 Has Zn-Dependent Nuclease Activity against E. coli rRNAs RNA degradation is observed as a decrease in the amount of 16 S and 23 S rRNAs with 1 μg of RNA used per lane. Control lanes contained either RNA with buffer or BSA instead of MTH1203, where the BSA concentration was equal to the highest concentration of MTH1203 in each gel. (A) Degradation assays performed at 37°C with increasing amounts of MTH1203. (B) Degradation assays performed at 37°C with different incubation times (15 μg of MTH1203 per lane). (C) Degradation assays performed at different temperatures (10 μg of MTH1203 per lane). (D) Inhibition of the nuclease activity by incubation with increasing concentrations of TPEN (10 μg of MTH1203 per lane). See also Figure S5. Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 7 Dimerization of MTH1203 Is Conserved and Suggests a Model for Degradation (A) Dimer of MTH1203 superposed with those of its archaeal orthologs Mm0695 (PDB ID 2xr1) and PH1404 (PDB ID 3af5). (B) Model of a single-stranded RNA binding to the MTH1203 dimer. Domains are colored as in Figure 2. See also Figures S4 and S5. Structure 2011 19, 622-632DOI: (10.1016/j.str.2011.03.002) Copyright © 2011 Elsevier Ltd Terms and Conditions